Afferents to the nucleus tractus solitarius utilizing excitatory amino acid transmitters were described in rat brain by autoradiography following microinfusion and retrograde transport of D-[3H]aspartate. Parallel experiments with the injection of [3H]GABA were employed to establish the transmitter-selective nature of the retrograde labelling found with D-[3H]aspartate. Following infusion of D-[3H]aspartate, perikaryal labelling was heaviest in myencephalon, where at least 16 discrete nuclei were labelled. Heaviest labelling was localized bilaterally in the trigeminal nucleus with cells extending through its subdivisions and the entire rostrocaudal axis. Intense labelling was also obtained in the inferior olive, predominantly contralaterally, and non-perikaryal labelling noted. Vestibular, reticular and raphe nuclei contained heavily labelled perikarya. In cervical spinal cord, a moderate density of labelled cells was found in dorsal horn, adjoining the central canal (lamina X) and in the central cervical nucleus, along with appreciable labelling of processes and non-perikaryal labelling. The relative density of labelled perikarya in mesencephalic nuclei was much lower than found in myencephalon, although D-[3H]aspartate produced topographic and precise labelling of a small number of cells in the periaqueductal gray, medial parabrachial nucleus and Koelliker-Fuse nucleus. Only weak labelling was found in cortex and hypothalamus. Labelled cells were not consistently observed in other regions (stria terminalis, amygdala, fastigial nucleus, locus coeruleus and rostral ventrolateral medulla) known to innervate the nucleus tractus solitarius. Lower densities of labelled perikarya were found after the microinjection of [3H]GABA, and the only regions in which a small number of cells were labelled by both D-[3H]aspartate and [3H]GABA were trigeminal nucleus, reticular nuclei and raphe obscurus. An exception was the ventrolateral medulla, where [3H]GABA produced precise labelling in the nucleus ambiguus and facial nucleus consistent with previous evidence for a GABAergic pathway from this area to the nucleus tractus solitarius. Our findings confirm the selectivity of the retrograde transport of D-[3H]aspartate and [3H]GABA. Overall, the transport of D-[3H]aspartate revealed a complex topographic and convergent network of afferent pathways to the nucleus tractus solitarius likely to utilize an excitatory amino acid transmitter.